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We explore how meeting members modify their responses to feedback according to the feedback modality and who receives the feedback. We conducted a field study and compared four feedback conditions: three using vibrotactile modality (chair vibration) and one using visual modality (spotlight flashing). The three vibrotactile conditions differ in the feedback recipients: potential speaker (a member whom other members would like to hear speak next, or a member who is willing to speak next), current speaker, and all members. Regarding the modality, the vibrotactile modality provided a moderate level of distraction of members (while the visual modality was low enough to be ignored) and led to more turn-taking than the visual modality. Regarding the recipients, members felt more positively about feedback when potential speaker, rather than current speaker, received feedback. Also members resulted in more turn-taking when all members or current speaker, rather than potential speaker, received feedback.

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the “footprints” that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.

This research was originally published in the Journal of Biological Chemistry. Shaoliang Li, Chisei Shimono, Naoko Norioka, Itsuko Nakano, Tetsuo Okubo, Yoshiko Yagi, Maria Hayashi, Yuya Sato, Hitomi Fujisaki, Shunji Hattori, Nobuo Sugiura, Koji Kimata and Kiyotoshi Sekiguchi. Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity. J. Biol. Chem. 2010; 285: 36645-36655 © the American Society for Biochemistry and Molecular Biology, Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys259-Lys263 and Lys270-Lys272, in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys259-Gly277 containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

In an attempt to elucidate the integrin-binding site within laminin-511 (alpha5beta1gamma1), we mapped the epitope for mAb 4C7, which recognizes the globular (G) domain of the laminin alpha5 chain and inhibits binding of integrin alpha6beta1 to laminin-511, using a series of recombinant laminin-511 mutants with deletions or substitutions in the G domain. Deletion of the LG2-5 modules only partially compromised the 4C7 binding activity, while deletion of all 5 LG modules completely abrogated the activity, indicating that the epitope for 4C7 resides in the LG1 module. In support of this conclusion, 4C7 reactivity was abolished when the LG1 module of laminin-511 was swapped with the corresponding module of laminin-111, but the reactivity was retained after swapping the LG2 or LG3 module. Despite the requirement of LG1 for 4C7 binding, a recombinant LG1 module failed to bind to 4C7 when expressed alone or in tandem with LG2, but exhibited significant 4C7 binding activity when expressed as an array of LG1-3. These results indicate that 4C7 recognizes an epitope in the LG1 module, whose active conformation is stabilized in the context of the LG1-3 modules. Despite their 4C7 binding activities, neither the recombinant LG1-3 fragment nor the LG2 and LG3 swap mutants were capable of binding to integrin alpha6beta1. Thus, the integrin binding activity does not necessarily parallel the 4C7 reactivity, and possibly requires a strictly defined conformation of the LG1 module which can only be attained within an array of the intact LG1-3 modules connected to the preceding coiled-coil domain.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.

Hydrolytic reactions in the presence of liposomes catalyzed by N ϵ -benzyloxycarbonylhistidine groups introduced into the side chains of poly[ N -(3-aminopropyl)glycine] were studied. On increasing the hydrophobicity of the polypeptide catalyst by introducing dodecyl groups into the side chains, and in the presence of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes, p -nitrophenyl palmitate (PNPP) was hydrolyzed more rapidly than p -nitrophenyl acetate (PNPA). The addition of cholesterol or phosphatidylserine to lipid bilayer membranes accelerated the hydrolysis of PNPP catalyzed by the polypeptide catalyst more strongly than that of PNPA. The substrate selectivity and catalytic efficiency of the polypeptide catalyst were found to be controlled by the physical state of the lipid bilayer membranes.

Poly[N-(3-aminopropyl)glycine] (PolyNAPGly, 5) and its derivatives were synthesized as model peptides of the membrane-bound enzymes, and investigated on their interactions with lipid membrane. PolyNAPGly (5) was shown to be distributed to dipalmitoylphosphatidyl choline bilayer membrane without destruction of the vesicle structure. Dodecyl groups were introduced to the side chains of PolyNAPGly (5), and found to facilitate binding of the polymer to lipid membrane. However, the introduction of the excess amount of hydrophobic groups to PolyNAPGly (5) caused the formation of polymer micelles, and the addition of such polymers to lipid vesicles destroyed the membrane structure. The derivative of PolyNAPGly (5) (n=35) bearing one or two dodecyl groups and some Ne-benzyloxycarbonylhistidyl groups was found to be bound tightly to lipid membrane without destruction of the membrane structure. The addition of these polypeptides to vesicles decreased the membrane fluidity.

Background Extended sitting time at work is viewed as a crucial public health issue. Encouraging workers to stand during their office hours via the installation of standing desks maybe one effective option to combat this. Here, we investigate whether the installation of high desks in the workplace can induce positive changes in the amount of physical activity (PA) and thereby lead to subsequent improvements in anthropometric parameters. Methods Thirty-two white-collar workers (22 men and 10 women, mean age 44.2) were randomly divided into two groups. A randomised crossover trial was performed for 13 weeks. During the experimental period, subjects completed their office work in a standing position using stationary high desks (standing work, SW) for 10 hours per week or more (SW period). The subjects were asked to maintain their normal sitting working habits during the control period (CONT period). The primary outcome was PA, which was assessed objectively using a triaxial accelerometer during weekdays and weekends. The secondary outcomes were anthropometric measurements. For each group and each parameter, the mean values during each period were recorded and were compared by paired t test. Results The daily total PA (10.2 ± 2.4 vs. 9.7 ± 2.3 METs · h/day, P = 0.043), MVPA (4.2 ± 2.2 vs. 3.7 ± 1.8 METs · h/day, P = 0.025), time spent in moderate PA (58.2 ± 20.7 vs. 53.4 ± 17.0 min/day, P = 0.019) and time spent in MVPA (62.8 ± 25.1 vs. 57.0 ± 20.3 min/day, P = 0.019) were significantly higher during the SW period compared to the CONT period. A weekdays verses weekends subanalysis revealed that these parameters were significantly higher during the SW period compared to the CONT period during weekdays only. No significant differences were noted before and after SW periods for most of the anthropometric measures, except waist circumference (83.7 ± 7.9 vs. 83.0 ± 7.9 cm, respectively, P = 0.007). Conclusions Standing work, via the installation of high desks, significantly increases moderate to vigorous physical activity, especially on weekdays. Trial registration UMIN-CRT, UMIN000016731, 7th March 2015.